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of 43 in PresS. Am J Physiol Heart Circ Physiol (March 30, 2007). doi:10.1152/ajpheart.01002.2006
Impairment of cardiac insulin signaling and myocardial contractile
performance in high cholesterol-fructose fed rats
Short running title: cardiac insulin resistance and contractile
dysfunction
Jen-Ying Deng,1 Jiung-Pang Huang,1 Long-Sheng Lu,2 and Li-Man Hung1, *
1
Department of Life Science, College of Medicine, Chang Gung University, Tao-Yuan,
Taiwan; and 2Institute of Pharmacology, College of Medicine, National Taiwan
University, Taipei, Taiwan.
*Correspondence:
Li-Man Hung, PhD
Department of Life Science, College of Medicine, Chang Gung University
No. 259, Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan
Tel: +886-3-2118800 ext 3338
Fax: +886-3-2118295
E-mail: [email protected]
Copyright Information
Copyright © 2007 by the American Physiological Society.
Page 2 of 43
Abstract
Although insulin resistance is recognized as a potent and prevalent risk factor
for coronary heart disease, less is known as to whether insulin resistance causes an
altered cardiac phenotype independent of coronary atherosclerosis. In this study, we
investigate the relationship between insulin resistance and cardiac contractile
dysfunctions by generating a new insulin resistance animal model with rats on high
cholesterol-fructose
diet.
Male
Sprague-Dawley
rats
were
given
high
cholesterol-fructose (HCF) diet for 15 weeks; the rats developed insulin resistance
syndrome characterized by elevated blood pressure, hyperlipidemia, hyperinsulinemia,
impaired glucose tolerance, and insulin resistance. The results show that HCF induced
insulin resistance not only in metabolic-response tissues (i.e. liver and muscle) but
also in the heart as well. Insulin-stimulated cardiac glucose uptake was significantly
reduced after 15 weeks of HCF feeding, and cardiac insulin resistance was associated
with blunted Akt-mediated insulin signaling along with GLUT4 (glucose transporter 4)
translocation. The basal FATP1 (fatty acid transporter 1) levels were increased in
HCF rat hearts. The cardiac performance of the HCF rats exhibited a marked
reduction in cardiac output, ejection fraction, stroke volume, and end-diastolic volume.
It also showed decreases in left ventricular end systolic elasticity, whereas the
effective arterial elasticity was increased. In addition, the relaxation time constant of
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Page 3 of 43
left ventricular pressure (tau) was prolonged in the HCF group. Overall, these results
indicate that insulin resistance reduction of cardiac glucose uptake is associated with
defects in insulin signaling. The cardiac metabolic alterations that impair contractile
functions may lead to the development of cardiomyopathy.
Keywords： insulin resistance; rat; Akt; glucose transporter; cardiac dysfunction; high
cholesterol-fructose diet
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Introduction
Heart disease is a leading cause of death in diabetic patients (43); with coronary
artery disease (CAD) and atherosclerosis being the primary reasons for increased
incidence of cardiovascular dysfunction (43, 38). However, a predisposition to heart
failure might also reflect the effects of underlying abnormalities in cardiac diastolic
function that can be detected in asymptomatic patients with diabetes (13, 4). Several
etiological factors have been put forward to explain why hyperglycemia and/or
diabetes tend to lead to diabetic cardiomyopathy. The accumulation of connective
tissues, insoluble collagens (1), and abnormalities of various proteins that regulate ion
flux (specifically intracellular calcium) (18), has been proposed as an explanation for
left ventricular wall stiffness and contractile dysfunctions. Recently, it has been
speculated that diabetic cardiomyopathy could also occur as a consequence of
metabolic alterations (7, 8, 23).
It is well known that under normal conditions, the adult heart utilizes
predominantly long chain fatty acids for most of its energy requirements (60–90%);
with glucose and lactate providing the rest (25). Since Randle et al. proposed the
existence of a glucose–fatty acid cycle in 1963 (33, 34), the link between glucose and
fatty acid metabolism has been widely accepted. Disruption of the balance between
glucose and fatty acid metabolism is often a primary defect observed in cardiac
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pathologies such as hypertrophy, heart failure, diabetes, dilated cardiomyopathy and
myocardial infarction (6, 11). Cardiac muscle is also a target of insulin (16);
impairment of insulin-stimulated cardiac glucose uptake has been described in animal
models of diabetes (12), obesity (15), and hypertension (27). Binding of insulin to its
receptor activates the tyrosine kinase activity of the receptor’s ß-subunit (22). This
leads to autophosphorylation as well as tyrosine phosphorylation of several insulin
receptors (IR) substrates. These substrates, in turn, interact with phosphatidylinositol
3-kinase (PI3K), and stimulates Akt, a downstream serine/threonine kinase which
induces glucose uptake via translocation of glucose transporter GLUT4 to the plasma
membrane (10). Abnormalities in insulin signaling account for insulin resistance.
Insulin resistance is an important risk factor for the development of hypertension,
atherosclerotic heart disease, left ventricular hypertrophy and dysfunction, and heart
failure (17, 19, 32). It reflects a disturbance of glucose metabolism and can potentially
worsen metabolic efficiency of both skeletal and cardiac muscles. The exact
mechanisms of cardiac insulin resistance on progression of left ventricular contractile
dysfunctions are not fully elucidated. In addition, there have been no studies of
cardiac dysfunction in type II diabetic rodent models other than genetically obese or
diabetic animals. The rodent model has the advantage of having atherosclerosis not
present to confuse the interpretation of the mechanism of diabetic cardiomyopathy.
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Therefore in this experiment, we have chosen the high cholesterol-fructose diet to
induce insulin resistance in rats and investigated whether insulin resistance has an
effect on cardiac insulin signaling and left ventricle contractile dysfunctions.
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Methods
Animals and diets
This investigation abides by the rules written in the Guide for the Care and Use
of Laboratory Animals, published by the US National Institutes of Health (NIH
publication No. 85-23, revised 1996).
Four week-old Sprague-Dawley (SD) rats
(body weights: 150 gm - 170 gm) were maintained in the Animal Center of Chang
Gung University, under an ambient temperature of 25 ± 1 ℃ and a light-dark period
of 12 hrs. The animals were maintained either on the chow diet (LabDiet® 5010)
containing 5.1% fat (linoleic acid, C18:2, unsaturated fatty acid), 23.5% protein, and
50.3% carbohydrate with water, or on a high-cholesterol diet (Harlan Teklad TD03468,
Indianapolis, Ind) with 10% fructose solution for 15 weeks. The high-cholesterol diet
contained 10.1% fat (5% of coconut oil and 5.1% linoleic acid), 17% protein, 51.6%
carbohydrate and 4% cholesterol. Both diets contained a standard mineral and vitamin
mixture. Body weight, water, and food intake were recorded weekly.
Biochemical analysis
Blood was collected from the femoral vein after pentobarbital (65 mg/kg, ip)
anesthesia for biochemical measurements. Plasma was used for the measurements of
total cholesterol, high-density lipoprotein, and triglyceride (RANDOX, UK). Insulin
was measured using a sandwich enzyme-linked immunosorbent assay (ELISA;
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Mercodia, Sweden). Insulin and glucose tolerance tests (ITT and GTT, respectively)
were performed on animals that had been fasted overnight. Animals were either
intraperitoneally injected with 1 unit/kg body weight of human regular insulin (Lilly)
or intravenously injected with 0.5 gm/kg body weight of glucose. Blood glucose
samples (0.2 ml for each time point) were collected from the femoral vein at 0, 5, 10,
20, 30, 60, 90, and 120 min after glucose administration and were determined by the
glucose oxidase method (Chemistry Analyzer; Auto analyzer Quik-Lab., Ames,
Spain).
Hemodynamic measurements
The animals were anesthetized with pentobarbital sodium (65 mg/kg ip) and
placed on controlled heating pads (TC-1000 Temperature Controller, CWE Inc. USA)
with the core temperature measured via a rectal probe maintained at 37°C. A microtip
pressure-volume catheter (SPR-838; Millar Instruments, Houston, TX) was inserted
into the right common carotid artery and advanced into the left ventricle (LV) under
pressure control as described (3, 30, 31). Polyethylene cannulas (PE-50) were inserted
into the right femoral artery for the measurement of mean arterial pressure (MAP).
After stabilizing for 20 min, the signals were continuously recorded at a sampling rate
of 1,000/s by using an ARIA pressure-volume (P-V) conductance system (Millar
Instruments) coupled to a Powerlab/4SP analog-to-digital converter (AD Instruments,
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Mountain View, CA). Data was displayed and recorded on a computer. All P-V loop
data were analyzed by using a cardiac P-V analysis program (PVAN3.2; Millar
Instruments), and the heart rate (HR), end-systolic volume (ESV), end-diastolic
volume (EDV), end-systolic pressure (ESP), end-diastolic pressure (EDP), stroke
volume (SV), ejection fraction (EF), cardiac output (CO), stroke work (SW), arterial
elastance (Ea; end-systolic pressure/SV), mean arterial pressure (MAP), maximal
slope of systolic pressure increment (max dP/dt), and diastolic decrement (min dP/dt)
were computed. The relaxation time constant (tau), an index of diastolic function, was
calculated by two different methods [Weiss method: regression of log (pressure)
versus time; Glantz method: regression of dP/dt vs. pressure] using PVAN3.2. Total
peripheral resistance (TPR) was calculated by the following equation: TPR=
MAP/CO. The hemodynamic parameters were also determined under conditions of
changing preload, elicited by transiently compressing the inferior vena cava (IVC)
using a cotton swab inserted through a small, transverse, upper abdominal incision.
This technique yields reproducible occlusions in animals without opening the chest
cavity. Because max dP/dt may be preload dependent, the P-V loops recorded at
different preloads were used to derive other useful systolic function indexes that may
be less influenced by loading conditions and cardiac mass. These measurements
include dP/dt-end diastolic volume (EDV) relation (dP/dt-EDV), end-systolic PV
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relation [maximum chamber elasticity (ESPVR), Emax], and the preload-recruitable
stroke work (PRSW), which represents the slope between SW and EDV and is
independent of chamber size and mass. The slope of the end-diastolic PV relationship
(EDPVR), an index of LV stiffness, was also calculated from P-V relations using
PVAN 3.2.
Immunoblotting
Tissue lysates (membranes and cytosolic fraction) were isolated from soleus
muscle, epididymal adipose, and cardiac tissues according to previously published
procedure with slight modifications (24). In brief, tissues were first homogenized in a
lysis buffer (M-PER; Pierce, USA) with 1 mM phenylmethylsulfonylfluoride (PMSF)
as a protease inhibitor. The tissue lysates were then ultracentrifuged at 50,000 rpm for
1 h at 4℃. The resulting supernatant was labeled as a cytosolic fraction. The resulting
pellet, which contained the crude membrane, was resuspended in M-PER (300~500 µl)
with 0.5% Triton X-100, incubated at 4℃ overnight, and centrifuged again at 15,700
g for 20 min. Finally, the supernatant was collected and labeled as a membrane
fraction. Protein samples of cytosolic and membrane lysates were subjected to 10%
SDS-PAGE and electrophoretically transferred to PVDF protein sequencing
membrane for 2 hrs. The membrane was blocked in 5% non-fat milk in Tris-buffered
saline with 0.1% Tween-20 (TBST). It was then washed and blotted with anti-GLUT1
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(Chemicon, USA), GLUT4 (Chemicon, USA), FATP1 (Santa Cruz, USA), and/or
CD36/FAT (Santa Cruz, USA) antibodies. Phosphorylation of Akt was detected with
anti-phospho-Akt (Ser473) and anti-phospho-Akt (Thr308) (Santa Cruz); Akt was
determined with anti-Akt antibody (Santa Cruz). The membrane was then incubated
with HRP-conjugated secondary antibody prior to chemiluminescence detection
(Pierce, USA).
Histology
Tissues were fixed overnight with 4% paraformaldehyde in PBS, dehydrated,
embedded in paraffin, sectioned (6–8 μm), and stained with haematoxylin and eosin.
Statistical analysis
Data were expressed as mean ± standard error (S.E.). The difference in body
weight, water intake, food intake, cholesterol, triglyceride, insulin, glucose tolerance
test, and insulin tolerance test were analyzed by two-way repeated-measured ANOVA;
others were analyzed by student t-test, and the significant difference was set at P<
0.05.
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Results
General characteristics in control and HCF rats
Male SD rats were fed with a high cholesterol diet with 10% fructose solution
for 15 weeks to induce insulin resistance. HCF rats developed insulin resistance
syndrome which was characterized by elevated blood pressure, an impaired glucose
tolerance during glucose challenge, as well as increased fasting plasma cholesterol,
triglyceride, and insulin. As shown in Fig. 1A and online Table 1, the rats fed with
high cholesterol-fructose diet gained slightly less weight than the control rats over the
study period. HCF rats also increased water intake and reduced food intake during the
experimental period as compared to the control rats (Fig. 1B and C). After 15 weeks
of feeding, there was no difference in fasting glucose levels and plasma HDL
(high-density lipoprotein) among the groups (see online supplement data, Table 1);
however, HCF rats showed higher levels of total plasma cholesterol, triglyceride, and
insulin than those of control rats (Fig. 1D, E, and F). In addition, HCF rats had an
increase in mean arterial pressure (MAP, 106.9±1.48 versus 127.8±1.51 mmHg, p<
0.001), systolic blood pressure (SBP, 122.1±1.96 versus 145.3±2.03 mmHg, p< 0.001),
and diastolic blood pressure (DBP, 105.3±1.55 versus 119.0±1.58 mmHg, p< 0.01).
Intravenous glucose tolerance test (IVGTT) was performed on rats that had
fasted overnight and had intravenously received bolus injections of glucose (500
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mg/kg) through the femoral vein. After glucose loading, the plasma glucose
concentration was elevated from 77.3±3.81 to 216.2±5.86 mg/dL (5 min after
administration, p<0.001), and then dropped to 80.2±2.13 mg/dL (2 hours after
administration, p<0.001; Fig. 2A) in the control rats. HCF impaired glucose tolerance
(Fig. 2A) and the efficiency of insulin responses in IVGTTs (Fig. 2B). In addition,
HCF also impaired insulin sensitivities in IPITTs (intraperitoneal insulin tolerance test)
as compared to the control rats (Fig. 2C).
Impaired insulin signaling in HCF rats
Expression of glucose transporter (GLUT1 and GLUT4) proteins were
examined by immunoblotting method in experimental rat soleus muscle and
epididymal fat pad after 15 weeks of high cholesterol-fructose diet. HCF rats had a
dramatic reduction of membranous GLUT4 protein levels in soleus muscle as
compared to the control rats (Fig. 3A). In contrast, there was no significant difference
in the GLUT1 protein levels between the two groups (Figure 3).
To confirm the effects of HCF on insulin-stimulated recruitment of GLUTs and
FATP to the cell surface of the heart, we subjected protein extracts from the heart to
Western blot analysis (Fig. 4). After insulin stimulation, the GLUT4 protein levels in
the membrane were 1.63 and 1.30 folds in control and HCF rats respectively (Fig. 4A
and 4C, p<0.05). The basal membranous fatty acid transporter 1 (FATP1) was
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significantly increased in the heart of HCF rats (p<0.05, Fig. 4E, F). However, the
membranous FATP1 and CD 36 protein levels were not affected by insulin
stimulation in both groups (Fig. 4E, F).
Insulin mediated phosphorylation of Akt was measured in the hearts of high
cholesterol-fructose diet fed rats to determine whether decreased cardiac insulin
signaling (insulin resistance) was responsible for impaired GLUT4 membrane
translocation. Intravenous injections of insulin significantly increased cardiac Akt
phosphorylation (residue serine 473 and threonine 308 of Akt) in control rats (Fig. 5).
Insulin increased cardiac Akt-ser473 phosphorylation in rats fed with chow and high
cholesterol-fructose by 1.51 and 1.67 fold respectively. Although insulin induced
Akt-ser473 phosphorylation in both diet groups, the insulin mediated induction in
cardiac Akt-thr308 phosphorylation was almost completely blocked in HCF rats
(insulin-induced Akt-thr308 phosphorylation by 1.43 and 0.98 fold in control and
HCF rats respectively, p<0.05, Fig. 5).
Attenuated cardiac contractile functions in HCF rats
Impaired insulin signaling may have divergent or distinct effects on the
progression of cardiomyopathy in rats fed with high cholesterol-fructose diet for 15
weeks. We sought to directly measure the cardiac performance by Millar
pressure-volume instruments. The hemodynamic parameters (cardiac output, stroke
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work, maximal power, ejection fraction, stroke volume, maximum volume,
end-diastolic volume, dV/dt max, and dV/dt min) were reduced significantly in HCF
rats as compared to the control rats (Fig. 6 and online supplement data, Table 2).
Animals fed with high cholesterol-fructose diet for 15 weeks also prolonged the
relaxation time constant of left ventricular pressure (tau, p<0.05, Fig. 6J) and
increased the effective arterial elasticity (p<0.01, Fig. 6K). Fig. 7 illustrates typical
P-V loops obtained after inferior vena cava occlusions in both groups. The slopes of
systolic P-V relations (ESPVR, Fig. 7A) were dramatically decreased in HCF rats as
compared to the control rats (p<0.001, Fig. 7B).
Altered heart morphology in HCF rats
After 15 weeks of HCF feeding, the appearances of HCF rat hearts were larger
than those of control rats (Fig. 8A, left panel). The hearts weighted 1.41±0.15 gm and
1.17±0.1 gm in control and HCF rats respectively (p<0.05, n=8). However, both
groups had identical weights when normalized to the body weight; 2.55±0.19 mg/g
and 2.58±0.10 mg/g for control and HCF group respectively (Fig. 8A, left panel).
Transverse sections of HCF hearts showed dilatation of ventricle chamber and
decrease in ventricular wall thickness (Fig. 8B). Hematoxylin- and eosin-stained
sections of HCF rat hearts presented an increase in distance between myocytes and
thinner cardiomyocytes as compared to the control group (Fig. 8C).
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Discussion
Both genetic and environmental factors contribute to the development of
metabolic abnormalities. Several experimental studies have demonstrated that the
macronutrient composition of a diet is an important environmental determinant of the
quality of insulin action (2, 5). High-fat and high-fructose intakes were shown to
contribute to conditions such as hyperlipidemia, glucose intolerance, hypertension,
and atherosclerosis (26, 39). In addition, brief feeding of excess atherogenic diet
(chow with 45% kcal from fat and 2% cholesterol) produces striking features of
metabolic syndrome and coronary artery disease (14). High sugar intake is linked to
an increased risk of heart diseases. Simple sugars are the primary source of high
triglycerides (a type of blood fat) and very low-density lipoproteins (LDL), which are
independent risk factors for atherosclerosis. Sugar lowers high-density lipoprotein
(HDL) cholesterol and raises LDL cholesterol along with blood pressure levels. In
addition, it has been suggested that fructose induced hyperuricemia results in
endothelial dysfunction and insulin resistance, and might be a causal mechanism of
the metabolic syndrome (28). In the present study, HCF rats also showed
hyperuricemia (0.62±0.06 versus 1.36±0.15 mg/dl, p< 0.001). Sugar sweetened
beverages in the market today contain 12-15% sucrose; this factor should not be
ignored in regards to the development of insulin resistance and cardiovascular
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diseases (CAD) in the population. Therefore, in this study we have chosen a high
cholesterol diet combination with 10% fructose in drinking water to investigate
whether diet-induced insulin resistance causes cardiac contractile dysfunctions. This
diet is relevant to human nutrition as it mimics a common Western diet with high
consumption of sugary drinks. The result shows that feeding with a high
cholesterol-fructose diet to SD rats resulted in a phenotype of insulin resistance
syndrome characterized by an increase in blood pressure, hyperlipidemia,
hyperinsulinemia, and insulin resistance. Body weight gain observed in rats fed with
high cholesterol-fructose diet was slightly lower than control rats over the study
period (Figure 1 and online supplement data, Table 1). This was due to HCF rats
having increase in water (10% fructose solution) intake along with a reduction in food
(high cholesterol diet) intake during the experimental period. As observed through the
calculation of the energy expenditure in the 15th week, the energy intakes did not
differ significantly between the two groups (121.8 and 120.43 kcal/ rat/ day in the
control
and
HCF
rats
respectively);
thus
the
animals
fed
with
high
cholesterol-fructose diet did not develop obesity. Although approximately 70 % of
individuals in insulin resistance were overweight/obese, 30 % of those were
underweight/lean (35). Obesity promotes states of both chronic low-grade
inflammation and insulin resistance. However, even in the absence of obesity,
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infusion of animals with inflammatory cytokines or lipids can cause insulin resistance
(42). Elevation of plasma triglycerides and reduction of HDL are frequently observed
in
patients
with
insulin
resistance
and/or
diabetes
(21).
Feeding
high
cholesterol-fructose diet to SD rats resulted in dramatic increases in plasma
cholesterol and triglyceride with slightly decreases in HDL (Figure 1 and online
supplement data, Table 1). Since elevation of plasma triglycerides in humans has been
associated with consumption of high-carbohydrate diets (20), intake of 10% fructose
could have been responsible for increases in plasma triglyceride levels in HCF rats.
Insulin is a potent anabolic hormone and is essential for tissue development,
growth, and maintenance of whole-body glucose homeostasis. Failure of the target
cells to respond to insulin stimulation (such as insulin resistance) is commonly
observed under acute stress conditions and in individuals with obesity, metabolic
syndrome, or diabetes (41). In the present study, HCF-induced insulin resistance in
skeletal muscles was associated with a significant decrease in membrane GLUT4
levels (Figure 3). According to our findings, GLUT1 and GLUT4 levels of adipose
tissues were not altered with high cholesterol-fructose feeding; this might be due to
HCF-induced insulin resistance without development of obesity (Figure 1 and online
supplement data). Interestingly, the HCF rats also developed defects in cardiac insulin
action associated with blunted Akt-Ser308 phosphorylation in the heart (Figure 4 and
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5). The results suggest that HCF was not only shown to develop insulin resistance in
metabolic-response tissues (i.e. liver and muscle) but also in the heart as well.
Furthermore, HCF decreased GLUT4 and increased FATP1 levels, which indicated
that cardiac glucose uptake was reduced whereas fatty acid uptake might have been
elevated. Transgenic over-expression of fatty acid transport protein 1 in the heart
caused lipotoxic cardiomyopathy; suggesting that increases in fatty acid supply to the
heart adversely affect cardiac contractile functions (9).
Recent findings indicate that the perturbation in cardiac energy metabolism and
insulin resistance are among the earliest diabetes-induced events in the myocardium,
preceding both functional and pathological changes (36, 40). Furthermore, studies
have found myocardial insulin resistance in advance dilated cardiomyopathy limits
both glucose uptake and oxidation, and impairs the heart's ability to generate much
needed adenosine triphosphate (37). In order to evaluate cardiac functions, the Millar
pressure-volume instrument was used to determine left ventricular contractile
functions. The data shows that rats fed with HCF diet resulted in left ventricular
contractile dysfunctions (Figure 6, 7 and online supplement data, Table 2). Under
conditions of changing preload, the slopes of systolic P-V relationship (ESPVR) were
significantly decreased in the HCF rats; this caused a dramatic reduction in the stroke
volume because end-diastolic volume was decreased. These findings indicate the
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important role of cardiac insulin resistance in the pathogenesis of heart contractile
dysfunctions in diabetes and/or metabolic syndrome individuals.
Cardiac insulin signal not only regulates metabolic energy homeostasis but also
generates signals for cardiac growth, programmed cell death, and programmed cell
survival as well. During insulin resistance or diabetes, the heart rapidly modifies its
energy metabolism, resulting in augmented fatty acid and decreased glucose
consumption. Accumulating evidence suggests that this alteration of cardiac
metabolism plays an important role in the development of cardiomyopathy (29). Our
results have demonstrated that cardiomyocytes were dramatically shrunken in HCF
hearts. The transverse sections also show ventricular dilation and decrease in
ventricular wall thickness. These results suggest that cardiac insulin resistance may
lead to the development of dilated cardiomyopathy.
Overall, the results indicate that high-cholesterol food and sugar-sweetened
beverage that lead to maladaptive metabolic processes may interfere with the action of
insulin and increase susceptibility for the development of cardiomyopathy.
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Acknowledgments
This work was supported by grants from Chang Gung Memorial Hospital
(CMRPD 150011) and National Science Council (NSC 94-2320-B-182-024) of Taiwan
to Dr. Li-Man Hung.
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Figure Legend
Fig. 1. General characteristics of rats fed with chow (control, open circle) and high
cholesterol-fructose diet (HCF, closed circle) for 15 weeks. The body weight (A),
water intake (B), food intake (C), fasting cholesterol (D), triglyceride (E), and insulin
(F) were examined in control and HCF rats. Data are expressed as means ± SE (n =
25-30), *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. MAP, mean arterial pressure;
SBP, systolic blood pressure; DBP, diastolic blood pressure.
Fig. 2.
Intravenous glucose tolerance tests (IVGTT, A and B) and intraperitoneal
insulin tolerance tests (IPITTs, C) in rats fed with chow (control, open circle) and
high cholesterol- fructose diet (HCF, closed circle) for 15 weeks. During IVGTTs,
plasma glucose (A) and insulin (B) increased significantly in high cholesterolfructose diet rats as compared to chow diet fed rats. C, HCF impaired insulin
sensitivity during IPITTs. Data are expressed as means ± SE (n = 8), *P < 0.05, **P <
0.01, ***P < 0.001 vs. control.
Fig. 3.
Glucose transporter (GLUT) protein levels of the skeletal muscles and
epididymal
adipose
tissues
in
rats
fed
with
chow
(control)
and
high
cholesterol-fructose (HCF) diet for 15 weeks. The cytosolic and membranous GLUT1
27
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Page 28 of 43
and GLUT4 protein levels were examined for observation of GLUT1 and GLUT4
trafficking in soleus muscles (A, B, C, D) and epididymal fat pad (D, E, F, G). Equal
amount of proteins were resolved on 10% SDS-PAGE and blotted with respective
GLUT1 & 4 antibodies. All blots were stripped and re-probed with an antibody to
GAPDH or Na+-K+ ATPase (bottom). C, D, G, H are densitometric measurements of
protein bands in A, B, E, F, respectively. All experiments were performed in
quintuplicate from five animals.
Fig. 4.
Cardiac glucose transporter 1, 4 (GLUT1 and 4), fatty acid transport protein
1 (FATP1), and CD 36 protein levels in rats fed with chow (control) and high
cholesterol-fructose diet (HCF) for 15 weeks. Heart tissues were harvested 5 minutes
after intravenous injection of 0.9% NaCl or insulin (10 unit of human regular insulin,
Lilly). The cytosolic and membranous GLUT1, GLUT4, membranous FATP1 and CD
36 protein levels were examined in the heart. Equal amounts of proteins were resolved
on 10% SDS-PAGE and blotted with respective GLUT1, GLUT4, FATP1, and CD 36
antibodies. All blots were stripped and re-probed with an antibody to GAPDH or
Na+-K+ ATPase (bottom). C, D, F are densitometric measurements of protein bands in
A, B, E, respectively. All experiments were performed in quadruplicate from four
animals.
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Page 29 of 43
Fig. 5.
Cardiac Akt, phosphor-Thr308-Akt, and phospho-Ser473-Akt protein levels
in rats fed with chow (control) and high cholesterol-fructose diet (HCF) for 15 weeks.
Heart tissues were harvested 5 minutes after intravenous injection of 0.9% NaCl or
insulin (10 unit of human regular insulin, Lilly). Western blots of protein from cardiac
tissues probed with antibodies recognizing phosphorylation of residue serine 473 and
threonine 308 of Akt protein and total Akt protein. B is densitometric measurements
of protein bands in A. All experiments were performed in quadruplicate from four
animals.
Fig. 6.
Hemodynamic parameters were measured by Millar pressure-volume
conductance catheter system in rats fed with chow (control) and high
cholesterol-fructose diet (HCF) for 15 weeks. The cardiac output (A), stroke work (B),
maximal power (C), ejection fraction (D), stroke volume (F), end-diastolic volume
(G), dV/dt max (H), and dV/dt min (I) were significantly reduced in HCF group. On
the other hand, the HCF rats showed significantly increased tau_w (J) and arterial
elastance (K). Graph shows the means ± SE of 8-9 independent experiments.
Fig. 7. A, Representative pressure-volume relations following inferior vena cava
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Page 30 of 43
occlusions in rats fed with chow (control) and high cholesterol-fructose diet (HCF) for
15 weeks. Note that the slopes of end-systolic and end-diastolic pressure-volume (P-V)
relations (ESPVR and EDPVR) indicate left ventricular (LV) contractility and
stiffness respectively. B, The end-systolic elastancity was reduced significantly in the
HCF groups. Graph shows the means ± SE of 8-9 independent experiments.
Fig. 8. Morphology and structure of the hearts of rats fed with chow (control) and
those with high cholesterol-fructose diet (HCF) for 15 weeks. A, Photograph of the
heart (left) and the ratio of heart weight (HW) to body weight (BW) (right) in control
and HCF rats. Data are expressed as means ± SE (n = 8). B, Transverse sections of
control and HCF rat hearts. C, Heart tissues stained with hematoxyilin-eosin
(magnification ×400).
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A
Control
Body weight (g)
600
HCF
500
400
300
* *
200
***
** **
*
** *
100
0
0
Water intake (ml/day)
B
3
6
9
12
15
12
15
120
100
***
80
**
*
60
*
40
*
20
0
0
C
3
6
9
Food intake (gm/day)
35
30
25
***
20
15
10
5
0
0
Figure 1
3
6
9
Time (week)
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12
15
Page 32 of 43
D
Control
Cholesterol (mg/dl)
600
HCF
400
***
200
0
0
E
3
6
9
12
15
Triglyceride (mg/dl)
150
***
**
*
100
*
50
0
0
3
6
9
12
F
***
4
Insulin (ng/l)
15
3
2
*
1
0
0
3
6
9
Time (week)
Figure 1
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12
15
A
Plasma glucose (mg/dl)
Page 33 of 43
Control
***
300
HCF
***
***
***
200
***
**
*
100
0
0
20
40
60
80
100
120
Plasma insulin (ng/l)
B
4
3
**
2
*
1
**
0
C
Plasma glucose (mg/dl)
0
20
40
60
80
100
150
*
100
*
**
*
50
0
0
20
40
60
80
100 120
Time (min)
Figure 2
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120
Page 34 of 43
A
B
Cytosol
Membrane
Glut 4
Glut 4
Glut 1
Glut 1
Na+-K+
ATPase
GAPDH
C
Control
HCF
D
50
50
0
0
Glut 4
Glut4 / GAPDH (%)
100
100
100
50
50
0
0
Glut 4
Glut 1
Figure 3
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Glut 1
Glut1 / GAPDH (%)
100
Glut1 / ATPase (%)
Glut4 / ATPase (%)
p<0.001
Page 35 of 43
E
F
Membrane
Cytosol
Glut 4
Glut 4
Glut 1
Glut 1
Na+-K+
ATPase
GAPDH
50
50
0
0
Glut 4
100
100
50
50
0
0
Glut 4
Glut 1
Figure 3
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Glut 1
Glut1 / GAPDH (%)
100
Glut1 / ATPase (%)
100
Glut4 / GAPDH (%)
H
Glut4 / ATPase (%)
G
C ontrol
HCF
Page 36 of 43
A
B
Membrane
Diet
Insulin
C
HCF
-
-
C
+
Cytosol
HCF
+.
C
HCF
-
-
C
+
HCF
+
Glut 4
Glut 4
Glut 1
Glut 1
Na+-K+
ATPase
Glut 4
Glut 4
Glut 1
p<0.001
150
100
100
50
50
0
0
100
100
50
50
0
0
Diet
C
HCF
C
HCF
C
HCF
C
HCF
C
HCF
C
HCF
C
HCF
C
HCF
Insulin
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+
+
Figure 4
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Glut1 / GAPDH (%)
150
Glut1 / ATPase (%)
Glut4 / ATPase (%)
p<0.001
D
Glut 1
Glut1 / GAPDH (%)
C
GAPDH
Page 37 of 43
E
Diet
Insulin
C
HCF
-
-
C
+
HCF
+
FATP1
CD36
Na+-K+
ATPase
FATP1
F
CD36
C o n tr o l
HCF
150
150
100
100
50
50
0
0
Diet
C
HCF
C
HCF
Insulin
-
-
+
+
C HCF
-
Figure 4
Copyright Information
-
C
HCF
+
+
CD36 / ATPase (%)
FATP1 / ATPase (%)
p<0.05
Page 38 of 43
A
Diet
Insulin
C
HCF
-
-
C
+
HCF
+
p-Akt (ser473)
p-Akt (thr308)
Akt 1/2
P-Akt (ser 473)
B
P-Akt (thr 308)
p<0.05
p<0.001
p<0.05
p<0.05
150
150
p<0.01
100
100
50
50
0
0
Diet
C
HCF
C
HCF
C
HCF
C
HCF
Insulin
-
-
+
+
-
-
+
+
Figure 5
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Akt (thr 308) / Akt (%)
Akt (ser473) / Akt (%)
Control
HCF
Page 39 of 43
A
150000
p<0.01
30000
Stroke Work
(mmHg*μL)
100000
50000
10000
0
D
Maximal Power
(mWatts)
0
p<0.01
p<0.01
Ejection Fraction (%)
C
100
Control
HCF
20000
0
200
p<0.001
75
50
25
0
Figure 6
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E
Stroke Volume (μl)
Cardiac Output (μL/min)
B
p<0.001
300
200
100
0
Page 40 of 43
F
G
p<0.05
p<0.05
400
300
200
100
500
400
300
200
100
5000
p<0.05
15
10
5
0
Arterial Elastance (Ea)
(mmHg/mL)
p<0.01
2500
0
10000
K
10000
5000
p<0.05
0
J
Tau_w (msec)
dVdt min (μL/sec)
p<0.001
7500
15000
0
0
I
dVdt max (mL/sec)
End-diastolic Volume
(μl)
Maximum Volume (μL)
500
Control
HCF
H
1.00
0.75
0.50
0.25
0.00
Figure 6
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Page 41 of 43
A
Control
HCF
ESPVR
Pressure (mmHg)
Pressure (mmHg)
ESPVR
EDPVR
EDPVR
Volume (μL)
Volume (μL)
Figure 7
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Page 42 of 43
Control
HCF
End-systolic Elastance
(Ees) (mmHg/μL)
B
1.5
p<0.001
1.0
0.5
0.0
Figure 7
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Page 43 of 43
A
Control
HCF
HCF
HW/BW (mg/g)
Control
3
2
1
0
B
Control
HCF
C
Control
HCF
Figure 8
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